CN100370968C - Quercetin long-acting liposome powder injection and preparation method thereof - Google Patents

Abstract

The invention relates to a quercetin long-acting liposome powder injection and a preparation method thereof. The powder injection is composed of quercetin, polyethylene glycol-phosphatidylethanolamine, lecithin, cholesterol and excipients; the preparation method of the powder injection comprises 5-15 parts of polyethylene glycol-phosphatidylethanolamine, lecithin 40-60 parts, 5-15 parts of cholesterol, 20-50 parts of quercetin, the components are all by weight, and the above-mentioned raw materials are dissolved in an organic solvent with a ratio of 1-4:1 of chloroform and methanol. The quercetin long-acting liposome solution is prepared by a thin-film ultrasonic method, and excipients are added to the solution to obtain the quercetin long-acting liposome powder injection after freeze-drying. The quercetin long-acting liposome powder injection prepared by optimizing the process parameters of the present invention can be fully dissolved in the solvent used for intravenous infusion, which improves the absorption of quercetin in the body, prolongs its blood circulation time in the body, and then Increased bioavailability of quercetin.

Description

Quercetin long-acting liposome powder injection and preparation method thereof

1. Technical field

The invention relates to a quercetin long-acting liposome powder injection and a preparation method thereof, more specifically, relates to a liposome surface modified by polyethylene glycol-phosphatidylethanolamine (PEG-PE). Long-acting liposome, a quercetin long-acting liposome powder injection in which bioactive quercetin is encapsulated in long-acting liposomes and a preparation method thereof.

2. Background technology

Quercetin (3,3,4,5,7-phentahydroxyflavone) and its derivatives are the most widely distributed flavonoids in the plant kingdom, widely present in edible vegetables and fruits such as mountain dregs, apples, onions, tea leaves, honey, grapes, etc. etc. (Frankel et al., 1993; Hertog et al., 1993, 1996) and a variety of Chinese herbal medicines such as cypress leaves, galangal, coltsfoot, Panax notoginseng, ginkgo, etc. (Formica JV et al., 1995). Quercetin also has the same basic structure as other flavonoids, which is composed of three ring-shaped molecules connected to hydroxyl groups. The chemical structural formula of quercetin is:

Quercetin has attracted much attention due to its abundant and easy-to-obtain resources, strong biological activity and wide range of pharmacological effects. Since the beginning of this century, scholars at home and abroad have done a lot of research work on the extraction, purification, structure determination, physical and chemical properties, synthetic derivatives, pharmacology and clinical aspects of quercetin. A variety of biological activities and pharmacological effects of quercetin have been confirmed, it has antioxidant activity (Hayek T et al., 1997; Chopra M et al., 2000), anticancer (VermaAK et al., 1988; Deschner EE et al., 1991; Pereira MA et al., 1996), anti-inflammation (Ferry DR et al., 1996), anti-aggregation (Pignatelli P et al., 2000), dilation of coronary vessels (Perez-Vizcaino F et al., 2002) and other effects.

However, because quercetin extracted from plants is insoluble in water and many medicinal solvents, it is difficult to prepare ideal medicinal preparations from quercetin, which hinders the clinical application of quercetin. In order to improve the water solubility of quercetin and improve its absorption in the body, researchers at home and abroad have adopted various methods, such as dissolving quercetin in dimethyl sulfoxide organic solvent for clinical research, but due to two Excessive doses of methyl sulfoxide can cause lysis of red blood cells in the human body, and it will be terminated after use due to an unpleasant smell (Muther RS et al., 1980; Marshall LF et al., 1984). As another example, the researchers also modified the chemical structure of quercetin appropriately, and prepared water-soluble derivatives of quercetin by introducing hydrophilic groups into its structure, so that its absorption, distribution, and The running processes such as metabolism and excretion can be improved, and its pharmacological effects can be exerted (P.J.Mulholland et al., 2001). However, the method of chemical modification affects the pharmacological effects of quercetin, and there are problems such as separation and purification in chemical modification. Therefore, there is no suitable and economical way to apply quercetin as a medicine in clinical and industrial production. There is an urgent need to change the dosage form of quercetin, and to select an ideal delivery carrier to overcome the defects of quercetin’s poor water solubility and pharmacokinetics, and to make effective drugs target the liver and intestines, and to No side effects such as allergic reactions. The inventor of the present invention has made the present invention aiming at this problem.

As we all know, liposome, as a targeted drug carrier, is a new dosage form of targeted drug delivery system. At the end of the 1960s, people such as Rahman (Rahman Y E, et al.Proc Soc Exp Biol Med, 1979,146 (4): 1173) first used liposomes as drug carriers. Because the liposome has a cell-like structure, it can change the distribution of the encapsulated drug in the body, so that the drug mainly accumulates in tissues and organs such as the liver, spleen, lung and bone marrow, thereby improving the efficacy of the drug, reducing the therapeutic dose of the drug, and reducing the toxicity of the drug. . Therefore liposome has shown its obvious superiority in the treatment of many diseases as the carrier of many classes of drugs.

Long-acting liposomes usually incorporate some degradable biomaterials on the surface of common liposomes, such as polyethylene glycol (PEG) lipid derivatives, Tween, Brij (Zhang Xiaobin, Hou Xinpul.2003, 12( 2): 71-75), etc., wherein the long-acting liposome prepared by lipid derivatives of polyethylene glycol (Alexander L.Klibanov, Kazuo Maruyama, et al., 1990, 268 (1): 235-237) , because of its raw materials are easy to get, moderate price, no immunotoxicity and other characteristics and has advantages, the PEG part containing PEG lipid derivatives in the composition of long-acting liposomes provides long-acting liposomes hydrophilic surface, reducing The interaction between plasma proteins and cell surface ligands is reduced, thereby reducing the uptake of the mononuclear phagocyte system (MPS), prolonging blood circulation time, and enabling long-acting liposomes to effectively reach the lesion site.

Liposomes can be obtained by high-pressure milk homogenization (Hamilton RL J, Goerke J, et al., 1980, 21 (5): 981-992), thin film ultrasonic method (Huang C, 1969, 8 (1): 344) , emulsification dispersion method (Szoke F J, Papahadjopoulos D, et al., 1978, 75 (9): 4194-4198) and other methods.

3. Contents of the invention

Aiming at the existing defects of quercetin and its water-soluble derivatives in drug research and application, the object of the present invention is to provide a quercetin long-acting liposome powder injection, the powder injection Not only can the biologically active quercetin be fully dissolved in the solvent used for intravenous infusion, thereby prolonging the blood circulation time of quercetin in the body, enabling quercetin to reach the lesion effectively and improving its in vivo Absorption; another object of the present invention is to provide a preparation method of the above-mentioned quercetin long-acting liposome powder injection, which not only improves the raw materials and dosage used, optimizes the preparation parameters, but also is easy to operate and is suitable for large-scale continuous production .

The basic idea of the present invention provides on the one hand a kind of quercetin which is composed of biologically active quercetin, polyethylene glycol-phosphatidylethanolamine (PEG-PE) of different molecular weights, lecithin, cholesterol and excipients Long-acting liposome powder injection; on the other hand provide a kind of common liposome surface that adopts the polyethylene glycol-phosphatidylethanolamine modification lecithin and the cholesterol composition of the different molecular mass of biodegradable biological material, obtain long-acting liposome Plastid, long-acting liposome encapsulates quercetin as a directional drug carrier for quercetin, and improves the raw materials used, dosage, and preparation parameters to prepare quercetin long-acting liposome powder injection. This method not only obtains quercetin long-acting liposomes with high encapsulation efficiency and narrower particle size distribution, but also improves the pharmacological effects of quercetin so that quercetin is easily dissolved in pharmaceutical solvents and prolongs its lifetime. The blood circulation time in the body, improving its absorption in the body, so as to develop the research and application of quercetin with biological activity in medicine.

The object of the present invention is achieved by the technical solution consisting of the following measures.

The quercetin long-acting liposome powder injection of the present invention comprises 20-50 parts of quercetin with biological activity, 5-15 parts of polyethylene glycol-phosphatidylethanolamine, 40-60 parts of lecithin, and 5-5 parts of cholesterol. 15 parts, and 0.1-5 parts of excipients, wherein the molecular weight of polyethylene glycol in polyethylene glycol-phosphatidylethanolamine is 2000-8000, and the ratio of lecithin to cholesterol is 3-12:1, The components are by weight.

The polyethylene glycol-phosphatidylethanolamine used in the present invention is self-made, and the synthetic method is according to literature: Alexander L.Klibanov, Kazuo Maruyama, et al., 1990,268 (1): 235-237 gains.

In the above scheme, when the molecular weight of polyethylene glycol in the polyethylene glycol-phosphatidylethanolamine used is 2000, the amount of polyethylene glycol-phosphatidylethanolamine added is 5-15 parts, preferably 10-15 parts.

In the above scheme, when the molecular weight of polyethylene glycol in the polyethylene glycol-phosphatidylethanolamine used is 4000, the amount of polyethylene glycol-phosphatidylethanolamine added is 5-15 parts, preferably 5-11 parts.

In the above scheme, when the molecular weight of polyethylene glycol in the polyethylene glycol-phosphatidylethanolamine used is 8000, the amount of polyethylene glycol-phosphatidylethanolamine added is 5-15 parts, preferably 8-10 parts.

In the above scheme, the excipient is sorbitol, or mannitol, or glucose, or sucrose, or trehalose.

Realize the preparation method of quercetin long-acting liposome powder injection described in any one of the above schemes, adopt thin film ultrasonic method to make, according to the present invention, modify lecithin and cholesterol with polyethylene glycol-phosphatidylethanolamine of different molecular weights Long-acting liposomes are obtained on the surface of the common liposomes formed, and the long-acting liposomes wrap quercetin with biological activity. The raw materials and consumption, process parameters and main process steps used in the method are as follows:

(1) 20-50 parts of quercetin, 5-15 parts of polyethylene glycol-phosphatidylethanolamine, 40-60 parts of lecithin, and 5-15 parts of cholesterol, of which polyethylene glycol-phosphatidylethanolamine contains polyethylene glycol The molecular mass of alcohol is 2000-8000, the ratio of lecithin to cholesterol is 3-12:1, the above raw materials are all solid, measured in milligrams or grams or kilograms, organic solvents chloroform and methanol are liquid, their volume The ratio is 1-4:1, measured in milliliters or liters;

Dissolve the measured quercetin, polyethylene glycol-phosphatidylethanolamine, lecithin, and cholesterol in the organic solvent of chloroform and methanol in the above ratio, so that the concentration of the obtained solution is 1mg/ml-3mg/ml ml, then placed in a closed bottle, stirred at room temperature until completely dissolved;

(2) Transfer the above-mentioned completely dissolved solution to a flask connected to a rotary evaporator. The flask is immersed in a constant temperature water bath with a temperature of 30°C-50°C, and then the organic solvent is removed by vacuum rotary evaporation to form a layer on the wall of the flask. uniform film;

(3) Put the flask formed with the film into a vacuum drying oven, and dry it at room temperature for 2 hours to further remove the residual organic solvent;

(4) Add sterilized water into the dried flask, completely immerse the film on the bottle wall, and ultrasonically disperse it with an ultrasonic breaker. The ultrasonic power is 100w-1000w, and the ultrasonic time is 0.5-3 hours, to obtain quercetin Long-acting liposome solution;

(5) Add excipients to the quercetin long-acting liposome solution, freeze-dry, and store at a low temperature of 4° C. to 8° C. and keep the lid sealed for future use.

In the above scheme, when the molecular mass of polyethylene glycol in the polyethylene glycol-phosphatidylethanolamine used is 2000, the amount of polyethylene glycol-phosphatidylethanolamine added is 5-15 parts, preferably 10-15 parts.

In the above scheme, when the molecular mass of polyethylene glycol in the polyethylene glycol-phosphatidylethanolamine used is 4000, the amount of polyethylene glycol-phosphatidylethanolamine added is 5-15 parts, preferably 5-11 parts.

In the above scheme, when the molecular mass of polyethylene glycol in the polyethylene glycol-phosphatidylethanolamine used is 8000, the amount of polyethylene glycol-phosphatidylethanolamine added is 5-15 parts, preferably 8-10 parts.

In the above scheme, the excipient is sorbitol, or mannitol, or glucose, or sucrose, or trehalose.

The molecular weight of polyethylene glycol in the polyethylene glycol-phosphatidylethanolamine that the present invention selects to modify common liposome surface is 2000-8000, because polyethylene glycol-phosphatidylethanolamine is straight-chain molecular structure, chain length increases with molecular weight. Adding polyethylene glycol-phosphatidylethanolamine to the liposome membrane when preparing long-acting liposomes can effectively prolong the blood circulation time of quercetin in the body and make quercetin last longer. Effective liposomes can effectively reach the lesion, improve its absorption and stability in the body, and improve the pharmacokinetics of quercetin.

When the polyethylene glycol molecular weight in polyethylene glycol-phosphatidylethanolamine is 2000, because the molecular weight is relatively small, the molecular chain length of the formed polyethylene glycol-phosphatidylethanolamine is relatively short, that is, it is necessary to increase the molecular weight of the polyethylene glycol-phosphatidylethanolamine. The consumption of ethylene glycol-phosphatidylethanolamine can guarantee that the liposome has long-acting effect in the preparation process. Therefore, when polyethylene glycol-phosphatidylethanolamine with a molecular mass of polyethylene glycol of 2000 is used, the amount added is 5-15 parts, preferably 10-15 parts.

When using polyethylene glycol-phosphatidylethanolamine, the molecular weight of polyethylene glycol is 4000, because its molecular weight is moderate, which is more conducive to the stability and long-term effect of liposomes, therefore, adding polyethylene glycol-phosphatidylethanolamine The amount of ethanolamine is 5-15 parts, preferably 5-11 parts.

When the molecular weight of polyethylene glycol in polyethylene glycol-phosphatidylethanolamine is 8000, the viscosity of polyethylene glycol-phosphatidylethanolamine increases with the increase of molecular weight, if the amount of polyethylene glycol-phosphatidylethanolamine Too large, due to its high viscosity, the film of liposomes prepared by rotary evaporation is uneven, and the size of liposome particles is uneven after ultrasound, which makes the stability of long-acting liposomes poor. On the contrary, such as polyethylene glycol-phospholipids The consumption reduction of acyl ethanolamine can affect the long-acting property of liposome again, therefore, when polyethylene glycol molecular weight is 8000, the amount that adds polyethylene glycol-phosphatidylethanolamine is 5-15 parts, is preferably 8 parts. -10 copies.

The mass ratio of lecithin used in the present invention and cholesterol has influence on liposome particle size, as can be seen from Table 1, when the weight ratio of lecithin and cholesterol is 3: 1 and 4: 1, the uniformity and encapsulation of liposome particle Sealing rate is better, so the weight ratio of lecithin and cholesterol is preferably 4:1.

Table 1 Lecithin and cholesterol ratio optimization table

Lecithin/cholesterol (weight ratio, g or kg) Particle size (nm) 3:1 123±30 4:1 103±25 6:1 153±40 8:1 185±41

The phase transition temperature of lecithin is 30°C-50°C. Adding cholesterol to lecithin can change the phase transition temperature of lecithin. The phase transition temperature can affect the formation of lipid film, and then affect the size and uniformity of liposome particles. , this temperature can be regulated by the constant temperature water bath temperature during rotary evaporation. When preparing quercetin long-acting liposome powder injection in the present invention, after adding lecithin, cholesterol, quercetin, polyethylene glycol-phosphatidylethanolamine, the temperature when preparing the lipid film is 30°C-50°C, A preferred temperature is 40°C. Cholesterol can regulate the fluidity of the lipid membrane, increase the fluidity of the membrane, and improve the stability and encapsulation efficiency of liposomes.

The ratio of organic solvent chloroform and methanol used in the present invention also has an impact on the liposome particle size. As can be seen from Table 2, the optimal ratio of organic solvent chloroform and methanol is 1: 1 and 3: 1, preferably 3: 1. Volume is measured in milliliters or liters.

Table 2 Chloroform (CHCl ₃ ) and methanol (CH ₃ OH) solvent optimization table

Chloroform/methanol (volume ratio ml or L) 1:1 2:1 3:1 4:1 Particle size (nm) 136±35 162±45 137±30 148±38

The time for the ultrasonic breaker used in the present invention to break and disperse the film is related to its power. Prolong the break time and increase the break power, the smaller the particle size of the liposomes prepared, but the ultrasonic break time is too long and the power is too large, which will destroy the lipid. The integrity of the body membrane, thereby affecting the encapsulation efficiency of the drug. Therefore, the ultrasonic crushing time selected for the preparation of quercetin liposome powder injection in the present invention is 0.5-3 hours, and the ultrasonic crushing power is 100-1000W, which can ensure better particle size and high encapsulation efficiency.

Sephadex G-75 Sephadex G-75 Sephadex G-75 from Amersia is used for chromatographic detection of dextran gel, eluted with double distilled water, monitored at 394nm and the elution peaks are collected, and the present invention is known from the detected results The encapsulation efficiency of the prepared quercetin long-acting liposome is 95%.

The quercetin long-acting liposome powder injection of the present invention and its preparation method have the following characteristics:

1. The quercetin long-acting liposome powder injection of the present invention, after quercetin is wrapped with a long-acting liposome, can be fully dissolved in a medicinal solvent for intravenous infusion, such as 5% glucose solution, normal saline .

2. The quercetin long-acting liposome powder injection of the present invention is dissolved in a medicinal solvent and instilled as an injection, which can not only improve its absorption in the body, but also prolong its blood circulation time in the body, thereby further improving the quality of quercetin. Pharmacokinetics of corticosteroids.

3. The quercetin long-acting liposome powder injection of the present invention can be stored for a long time at a low temperature of 4°C to 8°C, and has good stability, as shown in Table 3.

Table 3 Stability test data of quercetin long-acting liposome powder injection at low temperature 4-8°C

batch number Time (month) Exterior Microscope inspection Quercetin long-acting liposome 0 Yellow-green fluffy substance No crystallization after dissolution, no aggregation 1 Yellow-green fluffy substance No crystallization after dissolution, no aggregation 6 Yellow-green fluffy substance No crystallization after dissolution, no aggregation 12 Yellow-green fluffy substance No crystallization after dissolution, no aggregation

4. In the preparation method provided by the present invention, since the surface of common liposomes with lecithin and cholesterol components is modified with polyethylene glycol-phosphatidylethanolamines of different molecular weights, and the preparation parameters are optimized, a higher encapsulation The long-acting liposome drug carrier with high efficiency and narrow particle size distribution, the long-acting liposome carrier is used to encapsulate the biologically active quercetin, so as to improve the pharmacological effect of quercetin and make quercetin easy to dissolve in the drug Use solvents to prolong its circulation time in the body and improve its absorption in the body.

4. Description of drawings

Fig. 1 is the photogram of quercetin long-acting liposome transmission electron microscope of the present invention.

Fig. 2 is a schematic diagram of the distribution of quercetin long-acting liposome morphology and particle size curve of the present invention.

From the photo of Fig. 1 transmission electron microscope irradiation, it can be seen that the quercetin long-acting liposome membrane of the present invention is fingerprint-like, and the helical structure is clear; the particle size distribution of the quercetin long-acting liposome of the present invention can be known by the curve distribution of Fig. 2 The range is narrow, and the average particle size is 100±25nm.

5. Specific implementation

The following examples and biological activity experiments are further detailed descriptions of the present invention, but do not imply any limitation to the present invention.

1, the present invention to the acute toxicity test of mouse:

①Take 20 BABL/c mice, 6-8 weeks, half male and half female, fast for 12 hours, drink water normally, adopt tail vein administration, the maximum dose of tail vein administration is 40mg of quercetin /ml, each dose is 0.2ml, which is equivalent to 480 times the daily dose of human beings. The mice did not show obvious symptoms of poisoning and death after taking the medicine. After continuous observation for one week, all the animals survived, moved freely, had smooth hair, and had a normal diet. 1 week later, the animal was dissected, and the internal organs were observed with the naked eye and a microscope, and no obvious pathological changes appeared.

2. Pharmacokinetic experiment of the present invention:

①Take 20 BABL/c mice, 6 to 8 weeks old, half male and half male, and randomly divide them into the control group, namely the first group, and the experimental group, namely the second group, with 10 mice in each group:

The first group: the content of quercetin in the common liposome of quercetin is 5 mg/ml, and each mouse is dosed with 0.2 ml, administered once through the tail vein.

5 minutes, 15 minutes, 30 minutes, 60 minutes, 120 minutes, 180 minutes, and 240 minutes after administration of each mouse, eyeball blood was taken, anticoagulated with heparin, centrifuged to obtain plasma, and its internal organs such as heart, lung, liver , spleen, and kidney were taken out, homogenized, extracted, and HPLC was used to detect the concentration of quercetin and its metabolites at different times and its distribution in various organs.

The second group: the content of quercetin in the quercetin long-acting liposome is 5 mg/ml, and the dose is 0.2 ml for each mouse, and the tail vein is administered once.

5 minutes, 15 minutes, 30 minutes, 60 minutes, 120 minutes, 180 minutes, 240 minutes, 300 minutes, 360 minutes, 480 minutes, 720 minutes, 1440 minutes after the administration of each mouse to take eye blood, heparin anticoagulation, Plasma was collected by centrifugation, and internal organs such as heart, lung, liver, spleen, and kidney were taken out separately, homogenized, extracted, and the concentration of quercetin and its metabolites at different times and its distribution in various organs were detected by HPLC.

As can be seen from the results of HPLC detection of blood drug concentration in mice, the blood drug concentration of mice in the quercetin common liposome group reached the highest value within 15 minutes of administration. The blood concentration of quercetin long-acting liposome group mice reached the highest value within 4 hours. Namely, the long-acting liposome of quercetin added with polyethylene glycol-phosphatidylethanolamine significantly prolongs the circulation time of the drug in mice. Moreover, tests on various organs found that after the injection of quercetin long-acting liposome powder injection, the concentrations of quercetin and its metabolites in the liver and lung were the highest in tissues taken at the same time. Compartment model A two-compartment model was used.

Table 4 Determination results of blood drug concentration in mice

Quercetin Ordinary Liposomal Powder Injection time (min) Concentration C (mg/L) In(C) T1/2α(min) T1/2β(min) AUC(0-t)(mg/L*min) AUC(0-∞)(mg/L*min) Cmax(mg/L) 5 10.37 2.339 35.259 69.315 1090.142 1404.176 17.39 15 17.39 2.856 30 7 1.946 60 4.3 1.459 120 2.95 1.082 180 2.57 0.944 240 1.35 0.3 Quercetin long-acting liposome powder injection 5 4.85 1.579 69.315 69.315 13415.599 18993.337 20.39 15 3.53 1.261 30 3.32 1.2 60 5.84 1.765 120 12.62 2.535 180 15.62 2.749 240 20.39 3.015 300 17.3 2.851 360 13.2 2.58 480 8.26 2.111 720 5.23 1.654 1440 1.625 0.486

Pharmacological test and result of the present invention:

② Take 40 BABL/c mice, 6-8 weeks old, half male and half male, and randomly divide them into 5 groups, the first and second groups are the control group, and the third, fourth and fifth groups are the experimental groups , 5×10 ⁵ C26 colon cancer cell lines were planted in the armpit of the right forelimb of the mouse. When the tumor grew to 0.5×0.5cm in size, the pharmacological experiments of each group were as follows:

The first group: normal saline group, 0.2ml normal saline was given by tail vein twice a week.

The second group: the liposome group, the liposome concentration is 5mg/ml, each dose is 0.2ml, and the tail vein is administered twice a week.

The third group: the content of quercetin in the quercetin long-acting liposome is 0.05mg/ml, each dose is 0.2ml, and the tail vein is administered twice a week.

The fourth group: the content of quercetin in the quercetin long-acting liposome is 0.5mg/ml, each dose is 0.2ml, and the tail vein is administered twice a week.

The fifth group: the content of quercetin in the quercetin long-acting liposome is 5mg/ml, each dose is 0.2ml, and the tail vein is administered twice a week.

The above groups were administered continuously for 4 weeks, and the size of the tumor was measured every week. According to statistical data, in the treatment group, the concentration of quercetin was the most obvious in the treatment group of 0.05mg/ml dosage group. In the normal saline group, the tumor inhibition rate reached 50%. During the treatment, the weight, hair, movement and feeding of the mice in the treatment group were better than those in the normal saline control group, and the liver, lung, kidney, brain, and heart tissues of the mice in the treatment group were tested, and there was no pathological reaction.

From above result, can further draw beneficial effect of the present invention as follows:

In the present invention, quercetin with biological activity is wrapped with long-acting liposome carrier to prepare quercetin long-acting liposome powder injection. Solubility and stability of the pigment, and adding a certain proportion of polyethylene glycol-phosphatidylethanolamine in the composition of quercetin liposomes, its effect is to prevent the plasma protein from being adsorbed on the surface of the liposomes, if there is no In PEG-phosphatidylethanolamine quercetin liposomes, plasma proteins quickly adhere to the surface of the liposomes, stimulating the rapid clearance of liposomes from the blood circulation by the mononuclear macrophage system. Liposomes containing polyethylene glycol-phosphatidylethanolamine can largely prevent adsorption and prolong blood circulation time, so drug-loaded liposomes can effectively reach lesion sites. In vivo cancer growth sites, infection and inflammation sites, and increased capillary permeability caused by lesions, long-acting liposomes containing quercetin can increase the aggregation at these sites, while the complete capillaries of normal tissues make large Some liposomes are impermeable. The quercetin liposome in the lesion, because the slow release of the drug directly acts on the lesion, increases the therapeutic effect, that is, in order to realize the active targeting of the drug, prolong the circulation time of quercetin in the body, improve the Bioavailability of quercetin.

The main instruments used in the preparation of the quercetin long-acting liposome powder injection in the present invention include a rotary evaporator, an ultrasonic breaker, a vacuum dryer, and a freeze dryer.

Example 1

Dissolve 60mg of lecithin, 6mg of cholesterol, 12mg of polyethylene glycol-phosphatidylethanolamine, the molecular weight of polyethylene glycol in polyethylene glycol-phosphatidylethanolamine is 2000, and 20mg of quercetin are dissolved in chloroform and methanol In 100ml of organic solvent, including 75ml of chloroform and 25ml of methanol, stir in a closed bottle until lecithin, cholesterol, polyethylene glycol-phosphatidylethanolamine and quercetin are completely dissolved, and place the completely dissolved solution in 250ml Connect the flask to a rotary evaporator, immerse the flask in a constant temperature water bath at 40°C, vacuum the rotary evaporation to remove the organic solvent, and form a uniform film on the wall of the flask, then put it into a vacuum drying oven, Dry at room temperature for 2 hours, add 100ml of sterilized water, ultrasonically disperse with an ultrasonic breaker, the power of the ultrasonic instrument is 200W, and the ultrasonic time is 1 hour, dissolve 1 mg of sorbitol in quercetin long-acting liposomes solution, and then placed in a lyophilizer to form a lyophilized powder, which is yellow-green flocculent quercetin liposome powder injection. Vacuum it or fill it with an inert gas to seal it, and store it at a temperature of 4°C-8°C for future use.

Example 2

0.50g lecithin, 0.10g cholesterol, 0.09g polyethylene glycol-phosphatidylethanolamine, the molecular weight of polyethylene glycol in polyethylene glycol-phosphatidylethanolamine is 3000, and 0.30g quercetin is dissolved in trichloro In 1L organic solvent of methane and methanol, the volume ratio of chloroform and methanol is v:v=1:1, stir in a closed bottle until lecithin, cholesterol, polyethylene glycol-phosphatidylethanolamine and quercetin Completely dissolve, put the completely dissolved solution in a 2.5L eggplant-shaped flask, connect the eggplant-shaped flask to a rotary evaporator, and immerse the flask in a constant temperature water bath at 30°C, vacuumize the rotary evaporation to remove the organic solvent, and form a circle on the wall of the flask Layer a uniform lipid film, then put it into a vacuum drying oven, dry it at room temperature for 2 hours, add 1L of sterilized water, and ultrasonically disperse it with an ultrasonic breaker, the ultrasonic power is 800W, and the ultrasonic time is 2 hours , dissolving 0.005g of sorbitol in quercetin long-acting liposome solution, and then placing it in a lyophilizer to form a freeze-dried powder, which is yellow-green flocculent quercetin liposome powder injection. Vacuum it or fill it with an inert gas to seal it, and store it at a temperature of 4°C-8°C for future use.

Example 3

Dissolve 45mg of lecithin, 8mg of cholesterol, 8mg of polyethylene glycol-phosphatidylethanolamine, the molecular weight of polyethylene glycol in polyethylene glycol-phosphatidylethanolamine is 2000, and 35mg of quercetin are dissolved in chloroform and methanol 200ml In the organic solvent, the volume ratio of chloroform and methanol is v:v=4:1, and the volume is measured in milliliters or liters. Stir in a closed bottle until lecithin, cholesterol, polyethylene glycol-phosphatidylethanolamine Dissolve completely with quercetin, place the dissolved solution in a 500ml eggplant-shaped flask, connect the eggplant-shaped flask to a rotary evaporator, and immerse the flask in a constant temperature water bath at 35°C, and vacuum the rotary evaporation to remove the organic solvent. Form a uniform film on the surface, then put it into a vacuum drying oven, dry it at room temperature for 2 hours, add 200ml of sterilized water, and carry out ultrasonic dispersion with an ultrasonic breaker, the ultrasonic power is 600W, and the ultrasonic time is 1 Hours, then 4 mg of mannitol was dissolved in the quercetin long-acting liposome solution, and then it was placed in a lyophilizer to form a freeze-dried powder, which was yellow-green flocculent quercetin liposome powder injection. Vacuum it or fill it with an inert gas to seal it, and store it at a temperature of 4°C-8°C for future use.

Example 4

Dissolve 39.9mg of lecithin, 5mg of cholesterol, 5mg of polyethylene glycol-phosphatidylethanolamine, the molecular weight of polyethylene glycol in polyethylene glycol-phosphatidylethanolamine is 3000, and 50mg of quercetin in chloroform and methanol 250ml In organic solvents, the volume ratio of chloroform and methanol is v:v=2:1, and the volume is measured in milliliters or liters. Stir in a closed bottle until lecithin, cholesterol, polyethylene glycol-phosphatidyl Ethanolamine and quercetin are completely dissolved, the dissolved solution is placed in a 500ml eggplant-shaped flask, the eggplant-shaped flask is connected to a rotary evaporator, the flask is immersed in a constant temperature water bath at 50°C, and the organic solvent is removed by vacuum rotary evaporation. Form a uniform film on the wall, then put it into a vacuum drying oven, dry it at room temperature for 2 hours, add 250ml of sterilized water, and carry out ultrasonic dispersion with an ultrasonic breaker, the ultrasonic power is 400W, and the ultrasonic time is In 1 hour, 5 mg of sucrose was dissolved in the quercetin long-acting liposome solution, and then placed in a lyophilizer to form a freeze-dried powder, which was yellow-green flocculent quercetin liposome powder injection. Vacuum it or fill it with an inert gas to seal it, and store it at a temperature of 4°C-8°C for future use.

Example 5

40mg of lecithin, 10mg of cholesterol, 12mg of polyethylene glycol-phosphatidylethanolamine, the molecular weight of polyethylene glycol in polyethylene glycol-phosphatidylethanolamine is 4000, 35mg of quercetin dissolved in chloroform and methanol In 150ml of organic solvent, the volume ratio of chloroform and methanol is v:v=3:1, and the volume is measured in milliliters or liters. Stir in a closed bottle until lecithin, cholesterol, polyethylene glycol-phosphatidyl Ethanolamine and quercetin are completely dissolved, and the dissolved solution is placed in a 250ml eggplant-shaped flask, and the eggplant-shaped flask is connected to a rotary evaporator, and the flask is immersed in a constant temperature water bath at 45°C, and the organic solvent is removed by vacuum rotary evaporation. Form a layer of uniform film on the wall, then put it into a vacuum drying oven, dry it at room temperature for 2 hours, add 150ml of sterilized water, and carry out ultrasonic dispersion with an ultrasonic breaker, the ultrasonic power is 400W, and the ultrasonic time For 1.5 hours, 1 mg of glucose was dissolved in the quercetin long-acting liposome solution, and then placed in a lyophilizer to form a lyophilized powder, which was yellow-green flocculent quercetin liposome powder injection. Vacuum it or fill it with an inert gas to seal it, and store it at a temperature of 4°C-8°C for future use.

Example 6

0.60g lecithin, 0.08g cholesterol, 0.10g polyethylene glycol-phosphatidylethanolamine, the molecular weight of polyethylene glycol in polyethylene glycol-phosphatidylethanolamine is 5000, and 0.20g quercetin is dissolved in trichloro In 1L organic solvent of methane and methanol, the volume ratio of chloroform and methanol is v:v=1:1, the volume is measured in milliliters or liters, and stirred in a closed bottle until lecithin, cholesterol, polyethylene glycol Alcohol-phosphatidylethanolamine and quercetin are completely dissolved, and the dissolved solution is placed in a 2.5L eggplant-shaped flask. Organic solvent, form a uniform film on the wall of the flask, then put it into a vacuum drying oven, dry at room temperature for 2 hours, add 1L of sterilized water, and carry out ultrasonic dispersion with an ultrasonic breaker, the ultrasonic power is 600W, ultrasonic time is 2 hours, then dissolve 5mg trehalose in quercetin long-acting liposome solution, and then place it in a freeze dryer to form a freeze-dried powder, which is yellow-green flocculent quercetin Liposome powder injection. Vacuum it or fill it with an inert gas to seal it, and store it at a temperature of 4°C-8°C for future use.

Example 7

Dissolve 40mg of lecithin, 5mg of cholesterol, 5mg of polyethylene glycol-phosphatidylethanolamine, the molecular weight of polyethylene glycol in polyethylene glycol-phosphatidylethanolamine is 4000, and 49.5mg of quercetin in chloroform and methanol 200ml In organic solvents, the volume ratio of chloroform and methanol is v:v=4:1, and the volume is measured in milliliters or liters. Stir in a closed bottle until lecithin, cholesterol, polyethylene glycol-phosphatidyl Ethanolamine and quercetin are completely dissolved, and the dissolved solution is placed in a 500ml eggplant-shaped flask. Form a uniform film on the surface, then put it into a vacuum drying oven, dry it at room temperature for 2 hours, add 200ml of sterilized water, and carry out ultrasonic dispersion with an ultrasonic breaker, the ultrasonic power is 600W, and the ultrasonic time is 1 After 2 hours, 2 mg of mannitol was dissolved in the quercetin long-acting liposome solution, and then it was placed in a lyophilizer to form a lyophilized powder, which was yellow-green flocculent quercetin liposome powder injection. Vacuum it or fill it with an inert gas to seal it, and store it at a temperature of 4°C-8°C for future use.

Example 8

Dissolve 45mg of lecithin, 9mg of cholesterol, 5mg of polyethylene glycol-phosphatidylethanolamine, the molecular weight of polyethylene glycol in polyethylene glycol-phosphatidylethanolamine is 6000, and 40mg of quercetin are dissolved in chloroform and methanol 300ml In the organic solvent, the volume ratio of chloroform and methanol is v:v=2:1, and the volume is measured in milliliters or liters. Stir in a closed bottle until lecithin, cholesterol, polyethylene glycol-phosphatidylethanolamine Dissolve completely with quercetin, place the dissolved solution in a 500ml eggplant-shaped flask, connect the eggplant-shaped flask to a rotary evaporator, immerse the flask in a constant temperature water bath at 35°C, and vacuum the rotary evaporation to remove the organic solvent. Form a layer of uniform film, then put it into a vacuum drying oven, dry it at room temperature for 2 hours, add 300ml of sterilized water, and perform ultrasonic dispersion with an ultrasonic breaker, the ultrasonic power is 800W, and the ultrasonic time is 1 hour , and then 1 mg of trehalose was dissolved in quercetin long-acting liposome solution, and then placed in a lyophilizer to form a lyophilized powder, which was a yellow-green flocculent quercetin liposome powder injection. Vacuum it or fill it with an inert gas to seal it, and store it at a temperature of 4°C-8°C for future use.

Example 9

55mg of lecithin, 10mg of cholesterol, 5mg of polyethylene glycol-phosphatidylethanolamine, the molecular weight of polyethylene glycol in polyethylene glycol-phosphatidylethanolamine is 8000, 25mg of quercetin dissolved in chloroform and methanol In 100ml of organic solvent, the volume ratio of chloroform and methanol is v:v=3:1, and the volume is measured in milliliters or liters. Stir in a closed bottle until lecithin, cholesterol, polyethylene glycol-phosphatidyl Ethanolamine and quercetin are completely dissolved, and the dissolved solution is placed in a 100ml eggplant-shaped flask, and the eggplant-shaped flask is connected to a rotary evaporator, and the flask is immersed in a constant temperature water bath at 40°C, and the organic solvent is removed by vacuum rotary evaporation. Form a uniform film on the wall, then put it into a vacuum drying oven, dry it at room temperature for 2 hours, add 100ml of sterilized water, and carry out ultrasonic dispersion with an ultrasonic breaker, the ultrasonic power is 200W, and the ultrasonic time is After 2 hours, 4 mg of sucrose was dissolved in the quercetin long-acting liposome solution, and then placed in a lyophilizer to form a freeze-dried powder, which was yellow-green flocculent quercetin liposome powder injection. Vacuum it or fill it with an inert gas to seal it, and store it at a temperature of 4°C-8°C for future use.

Example 10

Dissolve 40mg of lecithin, 10mg of cholesterol, 14.8mg of polyethylene glycol-phosphatidylethanolamine, the molecular weight of polyethylene glycol in polyethylene glycol-phosphatidylethanolamine is 7000, and 35mg of quercetin in chloroform and methanol In 200ml of organic solvents, the volume ratio of chloroform and methanol is v:v=4:1, and the volume is measured in milliliters or liters. Stir in a closed bottle until lecithin, cholesterol, polyethylene glycol-phospholipids Acetyl ethanolamine and quercetin are completely dissolved, and the dissolved solution is placed in a 500ml eggplant-shaped flask, and the eggplant-shaped flask is connected with a rotary evaporator, and the flask is immersed in a constant temperature water bath at 40°C, and the organic solvent is removed by vacuum rotary evaporation. Form a uniform film on the wall of the flask, then put it into a vacuum drying oven, dry it at room temperature for 2 hours, add 200ml of sterilized water, and carry out ultrasonic dispersion with an ultrasonic breaker, the ultrasonic power is 700W, and the ultrasonic time After 1 hour, dissolve 3 mg of glucose in quercetin long-acting liposome solution, and then place it in a freeze dryer to form a freeze-dried powder, which is yellow-green flocculent quercetin liposome powder injection . Vacuum it or fill it with an inert gas to seal it, and store it at a temperature of 4°C-8°C for future use.

Example 11

0.40g lecithin, 0.05g cholesterol, 0.05g polyethylene glycol-phosphatidylethanolamine, the molecular weight of polyethylene glycol in polyethylene glycol-phosphatidylethanolamine is 8000, and 0.47g quercetin is dissolved in trichloro In 1L organic solvent of methane and methanol, the volume ratio of chloroform and methanol is v:v=1:1, the volume is measured in milliliters or liters, and stirred in a closed bottle until lecithin, cholesterol, polyethylene glycol Alcohol-phosphatidylethanolamine and quercetin are completely dissolved, and the dissolved solution is placed in a 2.5L eggplant-shaped flask, and the eggplant-shaped flask is connected to a rotary evaporator, and the flask is immersed in a constant temperature water bath at 50°C, and removed by vacuum rotary evaporation. Organic solvent, form a uniform film on the wall of the flask, then put it into a vacuum drying oven, dry at room temperature for 2 hours, add 1L of sterilized water, and carry out ultrasonic dispersion with an ultrasonic breaker, the ultrasonic power is 800W, ultrasonic time is 2 hours, then dissolve 0.001g trehalose in quercetin long-acting liposome solution, and then place it in a freeze dryer to form a freeze-dried powder, which is yellow-green flocculent quercetin Vegetarian liposome powder injection. Vacuum it or fill it with an inert gas to seal it, and store it at a temperature of 4°C-8°C for future use.

Example 12

39mg of lecithin, 5mg of cholesterol, 5mg of polyethylene glycol-phosphatidylethanolamine, the molecular weight of polyethylene glycol in polyethylene glycol-phosphatidylethanolamine is 8000, 50mg of quercetin dissolved in chloroform and methanol In 150ml of organic solvent, the volume ratio of chloroform and methanol is v:v=2:1, and the volume is measured in milliliters or liters. Stir in a closed bottle until lecithin, cholesterol, polyethylene glycol-phosphatidyl Ethanolamine and quercetin are completely dissolved, and the dissolved solution is placed in a 250ml eggplant-shaped flask. Form a uniform film on the surface, then put it into a vacuum drying oven, dry it at room temperature for 2 hours, add 150ml of sterilized water, and carry out ultrasonic dispersion with an ultrasonic breaker, the ultrasonic power is 400W, and the ultrasonic time is 1 hour, then 5 mg of sorbitol was dissolved in the quercetin long-acting liposome solution, and then it was placed in a lyophilizer to form a freeze-dried powder, which was a yellow-green flocculent quercetin liposome powder injection. Vacuum it or fill it with an inert gas to seal it, and store it at a temperature of 4°C-8°C for future use.

Claims (14)

1. A quercetin long-acting liposome powder injection, characterized in that it consists of 20-50 parts of quercetin with biological activity, 5-15 parts of polyethylene glycol-phosphatidylethanolamine, and 40-60 parts of lecithin parts, 5-15 parts of cholesterol and 0.1-5 parts of excipients, wherein the molecular mass of polyethylene glycol is 2000-8000, the ratio of lecithin to cholesterol is 3-12:1, and its components are all based on On a weight basis, the excipient is selected from sorbitol, or mannitol, or glucose, or sucrose, or trehalose. 2. according to the described powder injection of claim 1, it is characterized in that when the molecular weight of polyethylene glycol used is 2000, the amount of adding polyethylene glycol-phosphatidylethanolamine is 5-15 parts. 3. according to the described powder injection of claim 1, it is characterized in that when the molecular weight of polyethylene glycol used is 2000, the amount of adding polyethylene glycol-phosphatidylethanolamine is 10-15 parts. 4. according to the described powder injection of claim 1, it is characterized in that when the molecular weight of polyethylene glycol used is 4000, the amount of adding polyethylene glycol-phosphatidylethanolamine is 5-15 parts. 5. according to the described powder injection of claim 1, it is characterized in that when the molecular weight of polyethylene glycol used is 4000, the amount of adding polyethylene glycol-phosphatidylethanolamine is 5-11 parts. 6. according to the described powder injection of claim 1, it is characterized in that when the molecular weight of polyethylene glycol used is 8000, the amount of adding polyethylene glycol-phosphatidylethanolamine is 5-15 parts. 7. According to the powder injection according to claim 1, it is characterized in that when the molecular weight of polyethylene glycol used is 8000, the amount of adding polyethylene glycol-phosphatidylethanolamine is 8-10 parts. 8. The preparation method of quercetin long-acting liposome powder injection described in any one of claims 1-7, adopts thin film ultrasonic method, it is characterized in that: adopt the polyethylene glycol-phosphatidylethanolamine modification egg of different molecular mass The common liposome surface that phospholipid and cholesterol are formed obtains long-acting liposome, and long-acting liposome wraps quercetin with biological activity, and the raw material used in this method and consumption, process parameter and process step are as follows: (1) 20-50 parts of quercetin, 5-15 parts of polyethylene glycol-phosphatidylethanolamine, 40-60 parts of lecithin, 5-15 parts of cholesterol, wherein the ratio of lecithin to cholesterol is 3-12: 1. The molecular mass of polyethylene glycol used is 2000-8000. The above raw materials are all solid, measured in milligrams or grams or kilograms. The organic solvents chloroform and methanol are liquid, and their volume ratio is 1-4:1 , measured in milliliters or liters; Dissolve the measured quercetin, polyethylene glycol-phosphatidylethanolamine, lecithin, and cholesterol in the organic solvent of chloroform and methanol in the above ratio, so that the concentration of the obtained solution is 1mg/ml-3mg/ml ml, then placed in a closed bottle, stirred at room temperature until completely dissolved; (2) Transfer the above-mentioned completely dissolved solution to a flask connected to a rotary evaporator. The flask is immersed in a constant temperature water bath with a temperature of 30°C-50°C, and then the organic solvent is removed by vacuum rotary evaporation to form a layer on the wall of the flask. uniform film; (3) Put the flask formed with the film into a vacuum drying oven, and dry it at room temperature for 2 hours to further remove the residual organic solvent; (4) Add sterilized water into the dried flask, completely immerse the film on the bottle wall, and ultrasonically disperse it with an ultrasonic breaker. The ultrasonic power is 100w-1000w, and the ultrasonic time is 0.5-3 hours, to obtain quercetin Long-acting liposome solution; (5) Add an excipient to the quercetin long-acting liposome solution, freeze-dry it, store it at a low temperature of 4°C-8°C and keep it sealed for future use. The excipient is selected from sorbitol or manna Alcohol, or glucose, or sucrose, or trehalose. 9. According to the preparation method of powder injection according to claim 8, it is characterized in that when the molecular weight of polyethylene glycol used is 2000, the amount of adding polyethylene glycol-phosphatidylethanolamine is 5-15 parts. 10. According to the method for preparing powder injection according to claim 8, it is characterized in that when the molecular weight of polyethylene glycol used is 2000, the amount of polyethylene glycol-phosphatidylethanolamine added is 10-15 parts. 11. According to the preparation method of powder injection according to claim 8, it is characterized in that when the molecular weight of polyethylene glycol used is 4000, the amount of polyethylene glycol-phosphatidylethanolamine added is 5-15 parts. 12. According to the preparation method of powder injection according to claim 8, it is characterized in that when the molecular weight of polyethylene glycol used is 4000, the amount of polyethylene glycol-phosphatidylethanolamine added is 5-11 parts. 13. According to the preparation method of powder injection according to claim 8, it is characterized in that when the molecular weight of polyethylene glycol used is 8000, the amount of polyethylene glycol-phosphatidylethanolamine added is 5-15 parts. 14. According to the preparation method of powder injection according to claim 8, it is characterized in that when the molecular weight of polyethylene glycol used is 8000, the amount of polyethylene glycol-phosphatidylethanolamine added is 8-10 parts.

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